A well known, epidemiologically reproducible risk factor for human carcinomas is the long-term consumption of "red meat" of mammalian origin. Although multiple theories have attempted to explain this human-specific association, none have been conclusively proven. We used an improved method to survey common foods for free and glycosidically bound forms of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc), showing that it is highly and selectively enriched in red meat. The bound form of Neu5Gc is bioavailable, undergoing metabolic incorporation into human tissues, despite being a foreign antigen. Interactions of this antigen with circulating anti-Neu5Gc antibodies could potentially incite inflammation. Indeed, when human-like Neu5Gc-deficient mice were fed bioavailable Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Such mice are already prone to develop occasional tumors of the liver, an organ that can incorporate dietary Neu5Gc. Neu5Gc-deficient mice immunized against Neu5Gc and fed bioavailable Neu5Gc developed a much higher incidence of hepatocellular carcinomas, with evidence of Neu5Gc accumulation. Taken together, our data provide an unusual mechanistic explanation for the epidemiological association between red meat consumption and carcinoma risk. This mechanism might also contribute to other chronic inflammatory processes epidemiologically associated with red meat consumption.

Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of ␣2,8-linked polysialic acid, represents the major virulence factor. In some K1 strains, phase-variable O-acetylation of the capsular polysaccharide is observed, a modification that is catalyzed by the prophage-encoded O-acetyltransferase NeuO. Phase variation is mediated by changes in the number of heptanucleotide repeats within the 5 -coding region of neuO, and full-length translation is restricted to repeat numbers that are a multiple of three. To understand the biochemical basis of K1 capsule O-acetylation, NeuO encoded by alleles containing 0, 12, 24, and 36 repeats was expressed and purified to homogeneity via a C-terminal hexahistidine tag. All NeuO variants assembled into hexamers and were enzymatically active with a high substrate specificity toward polysialic acid with >14 residues. Remarkably, the catalytic efficiency (k cat /K m donor ) increased linearly with increasing numbers of repeats, revealing a new mechanism for modulating NeuO activity. Using homology modeling, we predicted a three-dimensional structure primarily composed of a left-handed parallel ␤-helix with one protruding loop. Two amino acids critical for catalytic activity were identified and corresponding alanine substitutions, H119A and W143A, resulted in a complete loss of activity without affecting the oligomerization state. Our results indicate that in NeuO typical features of an acetyltransferase of the left-handed ␤-helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.Escherichia coli K1 is one of the main organisms causing bacterial sepsis and meningitis during the neonatal period (1-3). The pathogenesis of E. coli meningitis is a complex process involving colonization of the gastrointestinal tract, intestinal translocation, bacteremia, passage of the blood-brain barrier, and invasion of the arachnoidal space (4). Cell culture experiments indicated that transfer across the blood-brain barrier is mediated by transcytosis through brain endothelial cells (5, 6) and that, in contrast to other bacterial agents causing meningitis, E. coli K1 invades meningioma cells directly, leading to rapid cell death before an inflammatory response can be induced (7). Despite advances in diagnostics and therapeutics of neonatal infections, E. coli neonatal meningitis is still characterized by high rates of mortality and a high incidence of permanent neurological sequelae in those that survive (8, 9).E. coli K1 is protected by a thick layer of capsular polysaccharide, which is the major virulence factor important for serum resistance and vital passage of the blood-brain barrier (5, 10, 11). The K1 capsule consists of polysialic acid (polySia) 2 formed by the most prevalent sialic acid of humans, 5-N-acetyl neuraminic acid (Neu5Ac). Up to 200 residues are joined in ␣2,8-glycosidic linkages, resulting in a linear homopolymer that is structurally iden...

Background: Pathways for turnover and degradation of the mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) are currently unknown. Results: Mammalian cells can degrade Neu5Gc by sequential conversion into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, following release of glycolate and glucosamine 6-phosphate. Conclusion: Basic N-acetylhexosamine pathways seem tolerant toward the N-glycolyl substituent. Significance: We elucidate the metabolic turnover of the diet-derived human xeno-autoantigen Neu5Gc.

Oligosialic and polysialic acids (oligo/polySia) are characterized by high structural diversity, because of different types of sialic acids and glycosidic linkages. Although several methods have been described for the analysis of oligo/polySia, only high-performance liquid chromatography (HPLC) analysis in conjunction with 1,2-diamino-4,5-methylenedioxybenzene labeling, fluorometric C7/C9 detection, Western blotting, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF-MS) of lactonized oligo/polySia species, require submicrogram amounts of analyte. Since these methods do not provide detailed structural information, this study is focused on the characterization of oligo/polySia by tandem mass spectrometry (MS/MS). MALDI-TOF-MS/MS and electrospray ionization tandem mass spectrometry (ESI-MS/MS), employing up to three cycles of ion isolation and fragmentation in an ion trap, have been used for the characterization of nonderivatized glycans, oligoSia species modified at their reducing or nonreducing ends, as well as partially O-acetylated oligoSia derivatives. The obtained spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. However, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-chained oligoSia and long polySia species. Furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues.